The present invention relates to a method for enhancing growth in fungi and, more particularly, to the use of Bcl-2 or portions thereof for improving biomass production, survival, longevity, stress resistance and pathogenicity of filamentous fungi grown in solid or liquid culture.
Filamentous Fungus
From economic point of view, fungi dominate biotechnology. They are used in a variety of major industrial processes such as food and drug production, industrial paper and pulp production, agriculture, bioremediation and others. Perhaps the most well known metabolites produced by fungi are the β-lactam antibiotics produced by Penicillium, Acremonium, and Aspergillus species. Despite their availability for over 50 years, β-lactams still command 50% of the world market for systemic antibiotics which, in 1998 totaled nearly 21.5 billion US dollars, making it one of the most important categories of pharmaceutical sales (Schmidt, F. R. In: Esser, K. and Bennett, J. W. eds. The Mycota. 2002; Vol X, H. D. Osiewacz ed. Industrial Applications. Springer, Berlin, pp. 69-92). Of these, the total sales volume (1998) of cephalosporins was about 7 billion US dollars and that of broad-spectrum penicillins nearly 4 billion dollars. Improved strains have been generated that produce up to 50 g/liter penicillin and up to 30 g/liter cephalosporin, representing 50,000 fold improvement compared to production rates of the original strains. For many years, however, no further improvements in antibiotic production (β-lactams and non-β-lactams) rates have been achieved, mostly due to bottlenecks related to fermentation performance.
Another important group of therapeutic compounds are immunosuppressants (Anke T. ed. 1997; Fungal Biotechnology. Chapman and Hall, London; Kurnsteiner, H. et al. In: Esser, K. and Bennett, J. W. eds. The Mycota. 2002 Vol X, H. D. Osiewacz ed. Industrial Applications. Springer, Berlin, pp. 129-156). The most widely used compound in this category is cyclosporin A from Tolypocladium sp. with annual sales of approximately 2 billion US dollars. Similar to other fungal metabolites, cyclosporin A is produced mainly in submerged fermentation, and improvements in production performance are greatly needed.
Organic acids are another category of compounds that are widely produced by fungi. Citric acid is by far the most important organic acid in production volume (900,000 tons in 2000) as well as knowledge available (Ruijter, G. J. G. et al. In: Esser, K. and Bennett, J. W. eds. The Mycota. 2002, Vol X, H. D. Osiewacz ed. Industrial Applications. Springer, Berlin, pp. 213-230). Citric acid is mostly produced in submerged fermentation by the fungus A. niger. It is the most widely used organic acid in foods, beverages, pharmaceuticals, and technical applications. A number of other organic acids are produced by A. niger and other Aspergillus species, including fumaric acid, lactic acid, and gluconic acid. A range of additional secondary metabolites is produce in fermentation by fungi including vitamins, flavors, carotenoids, gibberellic acid, and more (for reviews see Oseiwacz, H. D. ed. In Esser, K. and Bennett, J. W. eds. The Mycota, 2002, Vol X, Springer, Berlin).
Fungal enzymes are crucial to various processes of the food and paper industries, as well as for bioremediation (waste treatment). Examples are the industrially produced cell wall degrading enzymes such as the cellulases, pectinases and xylanases. These enzymes are used mainly in fruit juice clarification, enzymatic pulping, in the baking industry, for paper bleaching and as animal feed. Edible fungal products are yet another large market. In addition to edible mushrooms, fungi are used in the food industry e.g., in cheese (Penicillium roqueforti, P. camemberti) and tofu production, production of flavorings, and as a protein source. Quorn, a mycoprotein produced in fermentation by Fusarium venenatum (syn graminearum) strain A3/5 has an annual sales volume of 200 million US dollars. Thus, there is a constant demand for F. venenatum A3/5 strains having improved growth rate and better performance in liquid fermentation (Wiebe, M. G. et al. Microbiology 1994;140: 3015-3021; Simpson, D. R., et al. Mycol Res 1998;102: 221-227).
Another area where fungi are widely used is for biological control of agricultural pests. Fungal biopesticides include bionematocides, bioinsecticides, biofungicides and bioherbicides. Most biological control products use fungal spores as the active ingredient. However, efficient pest control almost always involves application of large quantities of the bio-pesticide, which, in turn requires extremely large numbers of spores. For example, effective weed control with the leading bioherbicide product Collego requires 1012 spores per acre (Watson, A. K., et al. In: Prusky, D., Freeman, S. and Dickman, B. M. eds. Colletotrichum: Host Specificity, Pathology and Host-Pathogen Interaction. 2000; APS Press, St. Paul, Minn.). Furthermore, fungal fermentation products are often temperature sensitive and need to be stored under carefully controlled conditions, making the efficiency of spore production in solid or liquid fermentations, and spore quality important constituents of the cost of these products.
The abovementioned examples demonstrate the great economical importance, and yet unexploited potential of fungi. Of the bottlenecks in many fermentation processes, biomass production and sustainability of the culture during fermentation are crucial. Therefore, enhancement of growth rate, extenuation of the vegetative growth phase in culture, enhanced spore production, and improved resistance to environmental stress etc., all are highly desired traits that have wide applications in better utilization of fungi.
Bcl-2
Bcl-2 is a member of a superfamily of genes important to the regulation of programmed cell death processes known as apoptosis. The apoptotic-suppressing Bcl-2 gene was discovered as a proto-oncogene found at the breakpoints of the T(14/18) chromosomal translocations in low-grade B-cell lymphomas (Gross, A., et al. Genes and Development 1999;13: 1899-1911). Members of the Bcl-2 superfamily possess up to four-conserved Bcl-2 homology (BH) domains designated BH1, BH2, BH3 and BH4, which correspond to α-helical segments (Reed, C. J. Oncogene 1998;17: 3225-3236). Pro apoptotic genes contain only 3 BH domains while antiapoptotic genes contain all four BH domains. Deletion and mutagenesis studies revealed that the BH3 domain, and the presence of an alpha-helical transmembrane domain at the N-terminus are critical in the pro-apoptotic members for both dimerization with the anti-apoptotic proteins and for induction of apoptosis (Chou, J. J. et al. Cell 1999;96: 615-624). Examples of genes belonging to the Bcl-2 superfamily include the mammalian bcl-2, bax, bcl-XL, bcl-xS, bad, bak, Al and Mcl-1 genes, CED-9 from C. elegans, the BHRF1 gene (derived from Epstein-Barr virus) and the LMW5-HL gene (derived from African Swine Fever virus) (Takayama et al. Experimental Medicine, 1995;13: 24-31).
At least three general functions for Bcl-2 and some of its anti-apoptotic homologues such as Bcl-XL have been identified: (a) dimerization with other Bcl-2 family members; (b) Binding to non-homologous signal proteins involved in signal transduction, such as the protein kinase Raf-1, the protein phosphatase calcineurin and the small GTPases R-Ras and H-Ras; and (c) Formation of ion-channels pores (Reed, C. J. Oncogene 1998;17: 3225-3236; Reed, C. J. Nature 1997a; 387: 773-776; Zamzami, N. et al. Oncogene 1998a; 16: 2265-2282). Several biological effects of Bcl-2 on intact cells have been observed: Bcl-2 might act on plasma membrane (prevention of phosphatidylserine activity), on the cellular redox potential (decrease in lipid peroxidation, inhibition of reactive oxygen species, increase in catalase and superoxide dismutase, elevated NAD-/NADH ratio), effects on proteases (inhibition of caspase 3 and 6). Other effects of Bcl-2 include effects on intracellular ions (prevention of cytoplasmic acidification, inhibition of Ca++ uptake into the nucleus and ER) and effects on mitochondria (inhibition of pre-apoptotic mitochondrial transmembrane potential disruption, prevention of Ca++ influx, prevention of cytochrome C outflow from the intermembrane space, etc.). One of the first common manifestations of the apoptotic process, irrespective of the cell type and the induction stimulus, is a disruption of the mitochondrial membrane function that marks the “point of no return” of the apoptotic process. Given the functional importance of the Bcl-2 gene and it's homologues in apoptosis control, the Bcl-2 superfamily constitute prime targets for therapeutic interventions on numerous disease states. For example, it has been proposed that agents based on the proapoptotic Bax and Bad BH3 domains may have therapeutic value in induction of apoptosis in certain cancers (Shangary, S. and Johnson, D. E, Biochemistry 2002, 41; 9485-95).
Generally, it is known in the art that overall identity and homology among the genes belonging to the Bcl-2 superfamily, and having similar function, is surprisingly low at the nucleic acid and amino acid sequence levels. For example, identity between Baxa and Bcl-2 is about 21% and similarity there between is about 43% at their amino acid sequence level (Yamamoto, “Intercellular Signal Transduction”, Experimental Medicine, supp., Adduce Co., Ltd.). Nonetheless, the ability of Bcl-2 superfamily proteins to regulate cell life and death is conserved across evolution. For example, the nematode Caenorhabditis elegans contains a Bcl-2 homologue, CED-9, that is essential for the viability of these animals: thus, expression of the human Bcl-2 protein in C. elegans can rescue ced-9-deficient worms (Vaux et al., 1992; Hengartner and Horvitz, 1994). The human Bcl-2 protein can also block apoptotic cell death in insect cells (Alnemri, E. S. et al. Proc. Natl. Acad. Sci. USA 1992;89: 7295-7299), and can protect some yeast mutants from death induced by oxidative injury (Kane, D. J. et al. Science 1993;262: 1274-1276). Similarly, cell death induced by UV-B irradiation was suppressed in transgenic plants expressing Bcl-XL (Mitsuhara, I. et al. Current Biology 1999;9: 775-778). This is most likely due to the high interspecies sequence homology of the anti- and pro-apoptotic BH domains, with BH1 and BH2 being anti-apoptotic in character, and BH3 clearly associated with induction of apoptosis (Zhang et al, J Bio Chem 2000; 275:27303-06).
Application of the potential benefits of enhanced vigor, longevity and stress resistance conferred by expression of anti-apoptotic genes of the Bcl-2 family, such as Bcl-2, Bcl-XL and CED-9 has been proposed for a variety of organisms. For example, Bilbao et al. (U.S. Pat. No. 6,436,393) disclose adenovirus vectors encoding human Bcl-2 for transformation and protection of mammalian cells and organs during cryopreservation and transplantation. Expression of the Bcl-2 gene, under control of the CMV promoter, in transformed cells or transgenic animals, led to enhanced resistance to both freezing and thawing stress. In another application, human Bcl-XL and nematode CED-9 genes were introduced into tobacco plants by Agrobacterium transformation (U.S. Pat. No. 6,310,272 to Ohasi et al.), producing transgenic tobacco plants exhibiting enhanced resistance to UV, superoxide and salt stress. Similarly, Dickman, et al. (Dickman et al. PNAS USA 2001, 98: 6957-62), also using Agrobacterium transformation, generated transgenic tobacco plants expressing the human Bcl-2, Bcl-XL, nematode CED-9 and baculovirus op-iap members of the Bcl-2 family, all of which demonstrated heritable resistance to several necrotrophic fungal phytopathogens and tomato spotted wilt virus. Transformation of filamentous fingi with genes of the Bcl-2 family has not been reported.
Genetic Manipulation in Fungi
Traditionally, industrially important fungal species, such as the abovementioned Penicillium, Fusarium, Tolypocladium, Trichoderma, and Aspergillus sp., have been subjected to ongoing selection in the interest of optimizing production of desired metabolites or metabolic processes. For example, Inoue et al. reported the isolation of a fungus “overproducing” Streptovaricin C (U.S. Pat. No. 5,266,484 to Inoue et al.). Similarly, efforts have been undertaken for direction of fungal evolution, and favor the appearance of desired traits: for example, Kaira et al. (U.S. Pat. No. 6,475,772) and Scheilenberger et al. (U.S. Pat. No. 6,365,410) combined manipulation of rate of mutagenesis with careful choice of selection conditions to enhance production of fermentation products such as biofungicides. However, such selection methods are complex, laborious and costly, and with often unpredictable results.
Transformation and expression in filamentous fungi involving homologous expression has also been reported. Examples of homologous expression in filamentous fungi include the complementation of N. crassa mutants lacking key biosynthetic pathways, the complementation of the auxotrophic markers trpC, and argB in A. nidulans and the transformation of A. nidulans to acetamide or acrylamide utilization by expression of the A. nidulans gene encoding acetamidase (see, for example, Stohl et al. PNAS USA 1983;80:1058-62 or Grant et al. Mol Cell Bio 1984;4:2041-51; for an exhaustive review of homologous transformation see Hynes, M J Exper Mycology 1986; 10:1-8). Beijeresbergen et al. (U.S. Pat. No. 6,255,115) disclose the transformation of filamentous fungi such as A. awamori with homologous genes (from Fusarium) using the Agrobacterium tumifaciens Ti plasmid, affording a method of producing recombinant mold strains free of bacterial DNA contamination.
Recently, a number of fungal species have also been transformed using heterologous prokaryotic and eukaryotic genes. Examples of heterologous expression in filamentous fungi include the expression of a bacterial phosphotransferase in N. crassa, Dictyostellium discoideum and Cephalosporium acremonium, and the high-yield production of melanin in Streptomyces transformed with bacterial tyrosinase (U.S. Pat. No. 5,814,495 to della-Cioppa et al.). Berka et al. describe vectors for expression and secretion of heterologous proteins in filamentous fungi, devoid of bacterial DNA (U.S. Pat. No. 6,171,817 to Berka et al.). Using the vectors described, the authors produced transgenic A. awamori and A. nidulans expressing and secreting biologically active bovine chymosin, A. niger glucosamylase, and M. miehei carboxyl protease. Fungal transformation with high copy number (U.S. Pat. No. 6,090,574 to Giuseppin et al.) and over-expression of transformed genes (U.S. Pat. No. 6,403,362 to Moriya et al.) have also been reported recently. Thus, heterologous and homologous genes have been manipulated for increased production of desired gene products in filamentous fungi. However, none of the abovementioned methods have attempted to provide improved yields and longevity of fungal species by engineered enhancement of growth rate, extenuation of the vegetative growth phase in culture, enhanced spore production and improved resistance to environmental stress with heterologous gene products effecting programmed cell death and apoptosis.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods and novel media for improving fungal growth processes such as vegetative development, fungal sporogenesis and sustainability in culture and storage as well as, fungal pathogenicity, using transformation and treatment of filamentous fungi with heterologous Bcl-2 sequences and proteins.